A randomized, double blind, placebo controlled clinical trial was conducted in the neonatal high care unit of Tygerberg Children's Hospital (TBCH) Cape Town, South Africa for the period July 2011 to August 2012. The primary objective of the study was to assess the effect of probiotics on the incidence of NEC in high risk infants born to HIV-positive and HIV-negative women.

Throughout the study period, the standard of care protocol consisted of one dose (5 drops) probiotic/placebo daily for 4 weeks (28 days). This provided the study group with L. rhamnosus GG (0.35 x 109 colony-forming units [CFU]) and B. infantis (0.35 x 109 CFU) daily. The control group received placebo consisting of medium chain triglyceride (MCT) oil. Supplementation of the probiotic/placebo was initiated when enteral feeds started. Probiotic/ placebo supplementation was delayed/ halted in the event of: the infants being nill per os (NPO); when a query Necrotizing Enterocolitis (NEC) was suspected the infant continued with treatment until a confirmed a positive diagnosis of NEC I was made through abdominal X-ray; if the infant remained a query NEC and was NPO the infant did not receive probiotics/ placebo until the enteral feeds were commenced again. Supplementation was discontinued when HIV-exposed infants had a positive polymerase chain reaction (PCR) result on day 14 of life.

All study participants received human breast milk. Both the probiotics and placebo were mixed with the mothers own breast milk or donor breast milk before administration via the orogastric tube or orally. The probiotic/ placebo was added to the breast milk by the researcher and two research assistants who were blinded and not involved in the routine care of the infants. Participants exited the study on day 28 after birth or upon discharge from the hospital.

Probiotics administration in reducing the incidence and severity of NEC (as per Bell's criteria19) in premature very-low birth weight infants that are exposed to HIV. [ Time Frame: Each infant received supplementation for 28 days ] [ Designated as safety issue: No ]

Lactobacillus rhamnosus GG and Bifidobacterium infantis have been used as probiotics to reduce the incidence of NEC. The Product that was used is Pro-B2, it contains L. rhamnosus GG and B. infantis. The product was supplied by C Pharm. The study group received L. rhamnosus GG and B. infantis for 4 weeks (28 days). The control group received a placebo oil preparation, administered as drops of the oil vehicle used in manufacturing the Lactobacillus rhamnosus GG and Bifidobacterium infantis suspensions. A daily dose of 5 drops of the probiotic/ placebo was administered to the infants. The probiotics/ placebo was administered by the research assistant or investigator who only followed the assigned randomization of groups. The standard feeding protocol of the ward applied during the study. The breast milk of HIV positive mothers were pasteurised according to ward protocol.

Secondary Outcome Measures:

The prebiotic quality known as oligosaccharides of pasteurised own mothers breast milk of HIV positive and HIV negative mothers [ Time Frame: 3 milk samples in 38 days ] [ Designated as safety issue: No ]

Breast milk samples of HIV positive and HIV negative breast milk was tested. All breast milk samples were raw untreated specimens therefore milk samples did not undergo any pasteurization procedures Breast milk was collected in sterilised containers with lids Each sample contained a volume of 5-10ml of breast milk for the analysis

Sample 1 The first milk sample was collected on day four of lactation Sample 2 The second milk sample was collected on the tenth day of lactation Sample 3 The third sample was collected on the final day of the study

Day 28 after birth and the end of the neonatal period If a mother was unavailable during the breast milk sample collection the breast milk samples were collected during the first available time All milk samples were collected by the investigator or research assistant who was standardized for all protocol procedures

Other Outcome Measures:

The association between the prebiotic quality of breast milk and the incidence of NEC in premature very-low birth weight infants [ Time Frame: 3 milk samples in 28 days ] [ Designated as safety issue: No ]

Lactobacillus rhamnosus GG and Bifidobacterium infantis have been used as probiotics to reduce the incidence of NEC. The Product that was used is Pro-B2, it contains L. rhamnosus GG and B. infantis. The product was supplied by C Pharm. The study group received L. rhamnosus GG and B. infantis for 4 weeks (28 days). The control group received a placebo oil preparation, administered as drops of the oil vehicle used in manufacturing the Lactobacillus rhamnosus GG and Bifidobacterium infantis suspensions. A daily dose of 5 drops of the probiotic/ placebo was administered to the infants. The probiotics/ placebo was administered by the research assistant or investigator who only followed the assigned randomization of groups. The standard feeding protocol of the ward applied during the study. The breast milk of HIV positive mothers were pasteurised according to ward protocol.

Weight gain for both probiotic exposed and unexposed premature very-low birth weight infants [ Time Frame: Weight daily. Head circumfernce and length weekly. total follow-up was 28 days ] [ Designated as safety issue: No ]

All study infants were weighed daily by the nursing staff. Weights were taken for study infants that were incubated and those that were not. Daily weights were recorded from each participant's medical file by the investigator or research assistant. Any drastic weight chances were queried and the measurement was repeated under supervision of the investigator or research-assistants.

Infants were measured on day one of the study thereafter the length and head circumference was measured weekly. Upon exiting the study (day 28), the length and head circumference was measured again. Therefore five measurements on days 1, 7, 14, 21, 28 were taken.

Consecutive HIV positive or negative mothers, who gave birth to a premature, very-low birth weight infant at Tygerberg Childrens Hospital (TBCH) and consented to participate in the study.

Only mothers who decided to breastfeed after counseling, regardless of their HIV status were included

All HIV positive mothers are counseled routinely at TBCH about the risks and benefits of breastfeeding and bottle-feeding relating to the prevention of mother to child transmission scheme.

HIV positive mothers that were on the PMTCT treatment regiment and if antiretroviral medication was prescribed. Mothers receiving Nevirapine and Zidovudine as well as those receiving highly active antiretroviral medication were included in the study.

Premature/ low birth weight infants

Admission or transfer's to wards G1, G2, J3 and G8 at TBCH

Patients who were transferred to the kangaroo mother care unit in TBCH

Birth weight <1 250g

Premature infant with a birth age of <34 weeks gestation

Male and female subjects

Patients that received own mother's breast milk

Patients that received donor breast milk due to the following circumstances:

If donor breast milk was given as a supplemental feed due to insufficient breast milk supply from the mother

When the mother was unavailable e.g. home visits over weekends, continuation of work

If the mother was ill and unable to breastfeed the infant e.g. ICU admission

Infants with major congenital malformations and/or developmental disabilities pertaining to the gastrointestinal tract

Infants with perinatal asphyxia

Infants with a positive diagnosis of a complex heart disease

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Please refer to this study by its ClinicalTrials.gov identifier: NCT01868737